Primary platelet aggregation is essential for normal hemostasis and also plays a role in thrombosis. The results of studies on platelet aggregation over the past two decades indicate that normal platelet aggregation depends on the binding of fibrinogen to platelet glycoprotein GPIIb-IIIa. GPIIb-IIIa is a membrane glycoprotein of platelet cells which belongs to class of extracellular receptors called Integrins. Patients having a deficiency in GPIIb-IIIa are observed to have prolonged bleeding times or bleeding diathesis due to platelet dysfunction.
The platelet aggregation process may be viewed as a series of necessary cellular events: 1) an agonist-induced activation of GPIIb-IIIa which results in exposure of the fibrinogen binding site; 2) binding of fibrinogen ligand to the exposed binding site of GPIIb-IIIa; and 3) post-occupancy events pursuant to ligand binding. All three steps are believed to be important to normal platelet aggregation. Thus, even when GPIIb-IIIa is present in the platelet membrane, dysfunction of any of the above steps may result in defective platelet aggregation.
Support for the above view of events leading to platelet aggregation is found in several recent studies. First, it is found that efficient binding of large GPIIb-IIIa ligands, such as fibrinogen or certain anti-GPIIb-IIIa antibodies, requires platelet activation by an agonist such as ADP or thrombin. See, e.g., Shattil et al., J. Biol. Chem., 260:11107 (1985). Additionally, it is found that small fibrinogen-mimetic ligands such as Arg-Gly-Asp (RGD)-containing peptides bind to GPIIb-IIIa independent of activation. See, e.g., Lam, Sc-t, et al., J. Biol. Chem., 262:947-950 (1987); Frelinger, A. L. III, et al., J. Biol. Chem., 263:12397-402 (1988). This has led to the hypothesis that the fibrinogen binding region of GPIIb-IIIa has only a limited accessibility to large ligands absent agonist-induced activation of the glycoprotein. Agonist-induced expression of the fibrinogen receptor is proposed to involve G protein-mediated activation of phospholipase C, followed by activation of protein kinase C. See Shattil, S. J., et al., J. Biol. Chem., 262:992-1000 (1987). The exact nature of the changes in GPIIb-IIIa that render it able to bind fibrinogen are presently undetermined.
Post-occupancy events involving the GPIIb-IIIa complex also appear to play a role in platelet aggregation. For example, when GPIIb-IIIa is bound to either fibrinogen or an RGD-containing peptide, it is observed that certain anti-GPIIb and anti-GPIIb antibodies are capable of detecting the corresponding conformation change in the GPIIb-IIIa complex. This result suggests that conformational changes in the receptor/ligand complex occur subsequent to binding. How such conformational changes influence the extent of platelet aggregation is still unknown. See, e.g., Frelinger et al., supra; Frelinger et al., J. Biol. Chem., 265:6346 (1990). Moreover, conformational changes in the fibrinogen ligand are induced upon binding which may augment its adhesive function. See, e.g., Zamarron et al., Blood, 74:208a (Suppl. 1) (1989).
The most profound defects in platelet aggregation occur in patients afflicted with the hereditary disorder Glanzmann's thrombasthenia. The platelets of most homozygotes having the classic form of this disease possess less than 10% GPIIb-IIIa. In these individuals the observed defects in platelet aggregation can simply be attributed to the lack of functional GPIIb-IIIa.
However, other patients afflicted with Glanzmann's thrombasthenia display variant forms of the disease in which near normal levels of GPIIb-IIIa are present in the platelets. Accordingly, these latter individuals may have defects involving the activation, ligand binding, and/or post-occupancy functions of GPIIb-IIIa. Such defects are likely attributable to one or more primary defects in the amino acid sequence of GPIIb-IIIa.
Platelet aggregation defects are also observed in a number of clinical settings. For instance, myeloproliferative disorders, drug administration, uremia and post-cardiopulmonary bypass often involve acquired platelet aggregation defects. Such defects are likely to be attributable to one or more of the above described processes of activation, ligand binding, and post-occupancy defects.
Previous methods for diagnosing platelet aggregation dysfunction, e.g., platelet aggregometry, have not afforded classification of the disorder in terms of the primary steps of activation, binding, and post-occupancy events. It is desirable to characterize the disorder in terms of one or more defects in such steps in order to more accurately diagnose a patient's condition. More accurate diagnoses are expected to lead to improved treatment programs for patients with platelet dysfunction. Improved definition of aggregation defects would also be of value in research settings, such as in characterizing the modes of action of proposed anti-platelet drugs.